Infection by human papillomaviruses (HPV) at specific epithelium cells to induce epithelial proliferations plays an important role for cervical carcinogenesis. About 99 percent of confirmed cervical cancer cases are found to be associated with HPV infection with biopsy-confirmed squamous intraepithelial lesions (SIL) or cervical intraepithelial neoplasia (CIN). The incidence of HPV infection, primarily transmitted through sexual contact, is highest among young women and about 20 million sexually active men and women worldwide are currently infected. Approximately 1% of the population has genital warts and 4% of women have cervical precancerous lesions, such low grade of squamous intraepithelial lesion (LSIL), high grade of squamous intraepithelial lesion (HSIL) or atypical squamous cells of undetermined significance (ASCUS).
The presence of these lesions, preferentially observed in women aged 35-40 yrs, are at high risk of progression toward invasive cervical cancer. It is generally thought that persistent infection of human papillomavirus (HPV) is essential for developing precancerous epithelial lesions. Infections of high-risk types of HPV in women with LSIL may or may not progress to HSIL. In fact, remission occurs in the majority of LSIL human subjects while some progress to HSIL. Although 99.7% of cervical cancers are HPV positive, integration of viral genome into the host genome is required to facilitate the necessary genes to express for developing into HSIL or cancer. In fact, only one in every 10 women with persistent HPV infection may develop into higher grades of CIN lesions, such as cervical intraepithelial neoplasia (CIN) grade 2 and grade 3 (CIN2, and CIN3, respectively), and a portion of these epithelial lesion cases may ultimately progress into cervical cancer.
In the past, screening for cervical cancer is based on conventional cytology by papanicolaou (Pap) smear and suspicious smears are followed up with colposcopy, and/or histological biopsy. The use of cytological screening leads to a remarkable reduction in the mortality of cervical cancer. However, due to subjective test criteria, drawbacks of pap smear tests include difficulty in obtaining samples, poor inter- and intra-observer agreement, a high rate of false negatives (up to 20%) and false positives, the requirements for specialized labs staffed with highly trained personnel, and inability to identify a large proportion of HPV-infected persons. More reproducible assays are needed to improve the current screening method to avoid unnecessary medical intervention and psychological distress for the affected women. The current cervical cytology screening has sensitivity ranged from 30% to 87% and specificity ranged from 86% to 100%.
Detecting HPV infection by nucleic acid methods, such as “DNA Hybrid Capture”, has been developed, but is not ideal, due to not only its high cost, assay operation procedures, the requirements for facility, equipment, and highly trained personnel, but also its very low positive predictive value to CIN. In addition, DNA testing could not differentiate the diagnosis of LSIL from HSIL, nor CIN lesions from non-transforming latent or remissive viral infection. What is needed is a low cost, simple, sensitive and specific assay that can be performed on routine practice of a clinical lab or doctor office and is capable of detecting early stage of epithelial lesions, distinguishing LSIL from HSIL, and predicting the risk of progression into cervical cancer. Assays like PreTect HPV-Proofer® for the detection of E6/E7 mRNA suggested equivalent sensitivity to HPV DNA testing with higher positive predictive value. However, there are limited reports showing direct detection of E6/E7 oncoproteins in situ.
Known protocols for producing monoclonal antibodies are generally unsuitable for the production of anti-HPV monoclonal antibodies and cannot be used in immunocytochemical diagnostic tests for screening general human population. This is because antibodies produced by these protocols will not necessarily react with the naturally occurring HPV viral proteins in infected human cells. In addition, the epitopes recognized by prior antibodies will not necessarily be those epitopes which are resistant to the harsh procedures involved in standard sampling, fixing and storing of clinical specimens.
Three problems exist such that there is no antibody available to do clinical HPV detection. One is that HPV proteins in clinical samples are present in very small quantities. Secondly, there are too many HPV types and most HPV types present in clinical samples are not known or systemically identified due to the lack of available antibodies. Third, HPV virus can not be cultured in labs by standard tissue culture techniques. Therefore, there are no avaialbe HPV proteins purified in large quantities as immunogens for generating anti-HPV antibodies, and there are no available HPV proteins or antibodies as binding agents for clinical HPV detection.
Only 15 out of more than 100 types of HPV infections are considered to have a high risk of developing into CIN or cervical cancer. Also, around 70% of cervical cancer cases and 50% of CIN 2 and CIN 3 cases are attributed to high risk HPV type-16 and HPV type-18 infections. However, some progressive cervical cancer cases are related to infection by low risk HPV types, while infection of some HPV types will never progress into cervical cancer. For those reasons, it is important to identify those HPV infections with particular oncogenic proteins expression rather than just identify high risk types of HPV infection. Thus, there is a need for identifying HPV oncoproteins as cervical cancer biomarkers to better predict the risk of developing HSIL or cervical cancer-related diseases.
The development of appropriate assays, such as HPV immunoassays, is needed for the detection of such HPV oncoproteins or biomarkers for cervical cancer. The presence of E6/E7 oncoproteins in CIN 2 and CIN3 lesions could be evidence to indicate a high risk of progression. However, there are limited antibodies available for the detection of E6/E7 oncoproteins in situ. Therefore, there is a need to develop antibodies and assays for detecting HPV oncoproteins as cervical cancer biomarkers and screening for invasive cervical cancer and/or the risk for malignant transformation into cervical cancer.